Bioactive coating composition and methods

ABSTRACT

The present invention provides a bioactive coating composition, method and devices for bodily fluid-contacting surfaces. The coating comprises a complex of Formula II: wherein R 1  is an C 1-18 alkyl or C 6-32 aryl group, each R 2  is independently selected from the group consisting of C 1-18 alkyl and C 6-32 aryl, R 3  is N or O, n is a number from 1 to 10, and x in a number from 1 to about 30, directly bound to a heparin-activity molecule via covalent bonding, with one or more bioactive molecules bound to the heparin-activity molecule. The bioactive molecule may be an adhesive molecule such as fibronectin, a growth factor such as basic fibroblast growth factor, or any other bioactive molecule that binds, by any mechanism, to a heparin-activity molecule.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part application of U.S.patent application Ser. No. 09/629,059, entitled Amphipathic Coating forModulating Cellular Adhesion Composition and Methods, to Paul O. Zamora,Ray Tsang and Shigemasa Osaki, filed on Jul. 31, 2000, which is acontinuation-in-part application of U.S. patent application Ser. No.09/399,119, entitled Non-Thrombogenic Coating Compositions and Methodsfor Using Same, to Ray Tsang and Shigemasa Osaki, filed on Sep. 20,1999, which is a continuation patent application of U.S. Pat. No.5,955,588, entitled Non-Thrombogenic Coating Compositions and Methodsfor Using Same, to Ray Tsang and Shigemasa Osaki, and the specificationof each of the foregoing is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention (Technical Field)

[0003] The present invention relates to coatings, methods of use ofcoating compositions, and coated contacting surfaces of medical devices,wherein the coating includes silyl-heparin with one or more bioactivemolecules bound to the heparin, which bioactive molecules includeadhesive molecules, such as fibronectin for promoting cellularattachment, growth factor molecules, such as basic fibroblast growthfactor for promoting cellular growth, and a variety of other therapeuticmolecules for effecting one or more therapeutic purposes.

[0004] 2. Background Art

[0005] Note that the following discussion refers to a number ofpublications by author(s) and year of publication, and that due torecent publication dates certain publications are not to be consideredas prior art vis-a-vis the present invention. Discussion of suchpublications herein is given for more complete background and is not tobe construed as an admission that such publications are prior art forpatentability determination purposes.

[0006] Heparin is naturally present in various tissues, including liverand lung, as well as the luminal surface of endothelial cells. It iscomposed of repeating units of D-glucuronic acid and D-glucosamine, bothsulfated, in a 1,4-α linkage. Heparin is an anticoagulant, and it hasbeen reported that on the surface of endothelial cells it minimizesfibrin accumulation. When administered as a parenteral drug, heparinactivates anti-thrombin III, which leads to inactivation of thrombin andultimately systemic inhibition of fibrin formation.

[0007] A number of medical devices that come in contact with blood havebeen coated with heparin with the goal of taking advantage of itsthrombo-resistant nature. Stents, catheters, oxygenator fibers, andcardiac bypass circuits are examples of medical devices that have beencoated with heparin (Niimi et al., Anesth Analg 89:573-9, 1999; Inui etal., Artif Organs, 23:1107-12, 1999). Various strategies have beendeveloped to attach heparin to medical polymer surfaces includingchemical conjugation (Siefert et al., J Biomater Sci Polym Ed, 7:277-87,1995), plasma glow discharge methods (Kim et al., Biomaterials,21:121-30, 2000), the combination of both, and hydrophobic interactionas described herein (U.S. Pat. No. 5,955,588).

[0008] Heparin has a number of other biological actions related to itssimilarity to heparan sulfate. In the extracellular matrix. In theextracellular matrix, heparin and its chemical relative heparan sulfateis complexed into a scaffolding onto which cells attach. Heparin alsobinds to fibronectin and other adhesive molecules. In addition,extracellular matrix heparan sulfate and heparin also act as reservoirsfor growth factors, not only binding growth factors but also protectingthem from protease degradation. Fibroblast growth factor (FGF),platelet-derived growth factor (PDGF), and bone morphogenic protein(BMP) are examples of growth factors that complex to heparin.

[0009] The ability of heparin to bind adhesive molecules and growthfactors has lead to a number of efforts to use heparin complexes toimprove implantable medical device surfaces by providing surfaces towhich cells can attach and migrate. Other researchers have exploreddirect coatings of fibronectin, and peptides and peptide mimeticsderived from fibronectin, with the goal of increasing cell attachment(Walluscheck et al., Eur J Vasc Endovasc Surg, 12:321-30, 1996; Boxus etal., J Bioorg Med Chem, 6:1577-95, 1998; Tweden et al., J. Heart ValveDis, 4 Suppl 1:S90-7, 1995). Vascular grafts, for example, would beimproved by a surface that supports the growth of endothelial cells.Current vascular grafts of polytetrafluoroethylene and polyethyleneterephthalate do not support endothelization, and consequently patientsmust be maintained on long-term ant-platelet therapy.

[0010] Fibronectins function as adhesive, ligand molecules interactingwith specific receptors on the cell surface. Cells types that attach tofibronectin include fibroblasts, endothelial cells, smooth muscle cells,osteoblasts, and chondrocytes.

[0011] Other investigators have used heparin and fibronectin complexesto provide cell adhesion to polymeric surfaces. For example,heparin-albumin conjugates have been immobilized on carbon dioxide gasplasma-treated polystyrene (Bos et al., J. Biomed Mater Res, 47:279-91,1999) and complexed to fibronectin. The fibronectin on these surfacesincreased the attachment of endothelial cells. Bos et al. (Tissue Eng4:267-79, 1998) reported that endothelial cells grew to confluency onCO₂ gas plasma-treated polystyrene coated with an albumin-heparinconjugate. ishihara et al. (J Biomed Mater Res, 50: 144-152, 2000)reported that a heparin-conjugated polystyrene promoted cell attachmentof fibroblasts, smooth muscle cell and endothelial cells. Thefibroblasts grown on heparin-conjugated polystyrene had growth rates atleast comparable to fibronectin-coated, gelatin-coated, or tissueculture-treated media.

[0012] A simple method of efficiently complexing fibronectin, otheradhesive molecules, growth factor molecules, and therapeutic molecules,including derivatives or mimics of the foregoing, to a heparin complexwould have wide applicability for attaching cells to prostheses,including vascular grafts, bone and cartilage Implants, nerve guides andthe like. Particularly needed is a method and composition permitting useof a wide variety of adhesive molecules, including fibronectin, lamininand the like, and growth factor molecules, including FGF, as part of acoating for implantable medical devices.

[0013] There further remains a need in the art for coating compositionsfor implantable medical devices that promote cellular attachment, andfurther wherein cellular attachment can be modulated by the quantity ofadhesive molecule, and which can be applied simply and easily with nospecialized equipment or techniques.

[0014] A primary object of the present invention is to provide a coatingcomposition for contacting surfaces of implantable medical devices,wherein the composition comprises a silyl-heparin-bioactive moleculecomplex, attached to the contacting surface by hydrophobic interaction.

[0015] A further object of the invention is to provide an amphipathicsilyl-heparin-fibronectin coating composition for contacting surfaces ofimplantable medical devices, which promotes cellular attachment.

[0016] A further object of the invention is to provide a coating thecomposition of which can be varied, such that absent an adhesivemolecule the coating inhibits fibrin deposition, but when the coatingincludes an adhesive molecule, the coating promotes cellular attachmentand cell growth.

[0017] A further object of the invention is to provide a coating thecomposition of which can be varied, such that in one embodiment theinvention provides a silyl-heparin-growth factor molecule composition,and in another embodiment the invention provides asilyl-heparin-therapeutic molecule composition.

[0018] A further object of the invention is to provide coatingcompositions utilizing fibronectin, derivations of fibronectin, peptidemimics of fibronectin, laminin, vitronectin, thrombospondin, gelatin,collagen and subtypes thereof, gelatin, polylysine, polyornithine, andother adhesive molecules or derivatives or mimics of other adhesivemolecules.

[0019] A further object of the invention is to provide coatingcompositions utilizing fibroblast growth factor, platelet-derived growthfactor, vascular endothelial growth factor, hepatocyte growth factor,placental growth factor, insulin-like growth factor, nerve growthfactors and neurotrophins, heparin-binding epidermal growth factor,transforming growth factor-β, bone morphogenetic protein 2, osteogenicprotein 1 and keratinocyte growth factor, and other growth factormolecules or derivatives or mimics of other growth factor molecules.

[0020] A further object of the present invention is to provide a costeffective and commercially feasible method for coating polymeric medicaldevices, including biodegradable medical devices, with a coatingcomprising a bioactive molecule.

[0021] A further object of the present invention is to provide a costeffective and commercially feasible method for coating polymeric medicaldevices, including biodegradable medical devices, with a coatingcomprising a silyl-heparin-bioactive molecule composition.

[0022] A primary advantage of the present invention is that it providesfor coating contacting surfaces of medical devices of complex geometriesand surfaces with a durable and low-cost coating that promotes thedesired biological or therapeutic effect, depending on the bioactivemolecule selected.

[0023] Another advantage of the present invention is that it provides amethod for determining the disassociation rate ofsilyl-heparin-bioactive molecule complexes from contacting surfaces by,in part, determining the number or silyl units per silyl moiety, or thenumber of silyl moieties per heparin molecule, or both.

[0024] Other objects, advantages and novel features, and further scopeof applicability of the present invention will be set forth in part inthe detailed description to follow, taken in conjunction with theaccompanying drawings, and in part will become apparent to those skilledin the art upon examination of the following, or may be learned bypractice of the invention. The objects and advantages of the inventionmay be realized and attained by means of the instrumentalities andcombinations particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which are incorporated into and form apart of the specification, illustrate several embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The drawings are only for the purpose ofillustrating a preferred embodiment of the invention and are not to beconstrued as limiting the invention. In the drawings:

[0026]FIG. 1 is a titration plot of the amount of fibronectin andsilyl-heparin needed to support cell attachment;

[0027]FIG. 2A and FIG. 2B depict the absorbance resulting from heparinand fibronectin added in serial doubling dilutions measured usingassays;

[0028]FIG. 3 depicts a comparison of the inhibition of basic fibroblastgrowth factor (bFGF) binding to heparin-agarose by heparin and varioussilyl-heparin complexes determined by immunochemical methods. The silylmoiety contained three silyl units, with between 5 and 20 silyl moietiesper heparin molecule;

[0029]FIG. 4A and FIG. 4B are plots of the bFGF concentration on signalstrength, using primary and secondary antibodies at a dilution of 1:500(A) and at a dilution of 1:250 (B). The data represents the average±S.D.;

[0030]FIG. 5 is a plot depicting the effect of complexing adhesivemolecules to silyl-heparin on subsequent binding of bFGF. All adhesivemolecules (fibronectin, fibrinogen and type IV collagen) were complexedto silyl-heparin at a concentration of 100 μg/mL with unbound materialrinsed prior to binding of bFGF. The data is the average ±S.D, with theasterisk indicating p<0.05 by Student's t-test;

[0031]FIG. 6 is a plot depicting the effect of FGF-silyl-heparin fromcoated sutures across a porous membrane on the growth of bovine aortaendothelial cells after 4 days as determined by crystal violet staining.Data is the average absorbance ±S.D., with the asterisk indicatingp<0.05 by Student's t-test;

[0032]FIG. 7 is a plot, averaged from four different implants, depictingthe diameter of granulation tissue surrounding suture implants with nocoating, silyl-heparin, or FGF-silyl-heparin; and

[0033]FIG. 8 is a plot of binding of vascular endothelial growth factor(VEGF) to silyl-heparin, detected using anti-VEFG antibodies.

DESCRIPTION OF THE PREFERRED EMBODIMENTS BEST MODES FOR CARRYING OUT THEINVENTION

[0034] The present invention relates to bioactive coating compositionscomprising a silyl-heparin-bioactive molecule complex, methods formaking and using the same, and devices including the same. The silylcomprises a hydrophobic silyl moiety of Formula I:

[0035] wherein R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group, each R₂ isindependently selected from the group consisting of C₁₋₁₈alkyl and C₆₋₃₂aryl, R₃ is N or 0, and n is a number from 1 to 10. X is from 1 to about30, such that from 1 to about 30 silyl moieties are covalently bound toa heparin molecule. One or more bioactive molecules, which may be thesame or different, are bound to the heparin molecule.

[0036] In one embodiment, the present invention provides bioactivemolecules that are adhesive molecules, thereby forming an amphiphaticcoating that resists fibrin accumulation and promotes cell attachment.In another embodiment, the present invention provides for bioactivemolecules that are growth factors molecules, thereby forming a coatingproviding for regional or localized delivery of the growth factormolecules. In yet another embodiment, the present invention provides forbioactive molecules that are therapeutic molecules, thereby forming acoating providing for regional or localized delivery of the therapeuticmolecules.

[0037] In another embodiment, the present invention provides medicaldevices with the contacting surface thereof coated with moieties ofFormula I, which moieties are covalently bonded to a heparin molecule,with one or more bioactive molecules bound to the heparin molecule. Theinvention further includes medical devices coated with moieties ofFormula I, which moieties are covalently bonded to a heparin molecule,but without a bioactive molecule bound to the heparin, wherein thecoating inhibits fibrin accumulation and cellular adhesion.

[0038] In another embodiment, the present invention provides afibrin-resistant medical device that promotes surface cellular adhesionwithin the body, the device having surfaces for contacting blood, cells,tissues, or other fluids. The blood- or cell-contacting surfaces havecoated thereon a coating composition including the moieties of FormulaI, which moieties are covalently bonded to a heparin molecule, with anadhesive molecule, including but not limited to fibronectin, complexedby affinity interaction to the heparin.

[0039] In another aspect, the invention provides a complex of FormulaII:

[0040] wherein R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group, each R₂ isindependently selected from the group consisting of C₁₋₁₈ alkyl andC₆₋₃₂ aryl, R₃ is N or O, n is a number from 1 to 10, and x is a numberfrom 1 to about 30.

[0041] These and other aspects of the present invention are describedfurther in the description and examples of the invention which follow.

[0042] Unless otherwise defined, all technical and scientific termsemployed herein have their conventional meaning in the art. As usedherein, the following terms have the meanings ascribed to them.

[0043] “Alkyl” refers to linear branched or cyclic, saturated orunsaturated C₁₋₁₈ hydrocarbons such as methyl, ethyl, ethenyl, propyl,propenyl, iso-propyl, butyl, iso-butyl, t-butyl, pentyl, cyclopentyl,hexyl, cyclohexyl, octyl, and the like.

[0044] “Aryl” refers to unsaturated C₆₋₃₂ hydrocarbon rings that may besubstituted from 1-5 times with alkyl, halo, or other aryl groups. Arylalso includes bicyclic aryl groups. Specific examples of aryl groupsinclude but are not limited to phenyl, benzyl, dimethyl phenyl, tolyl,methyl benzyl, dimethyl benzyl, trimethyl phenyl, ethyl phenyl, ethylbenzyl, and the like.

[0045] “Adhesive molecule” refers to molecules which promote cellularattachment, adhesion or growth, including fibronectin, laminin,vitronectin, thrombospondin, heparin-binding domains, and heparansulfate binding domains, as well as synthetic polymers of amino acidscontaining adhesive sequences derived from any of the foregoing. Thisincludes, without limitation, peptides or polypeptides containing theamino acids with the single letter codes RGD, IKVAV, YIGSR, and thelike. Adhesive molecules also include lectins that bind to heparin andcarbohydrate moieties on cell surfaces.

[0046] “Bioactive molecule” refers to any molecule with biologicalactivity within the body, including molecules used as a drug, to effecta biochemical change in an organism, or to confer a benefit to anorganism. The bioactive molecule preferably binds to one or more formsof heparin. A bioactive molecule includes an adhesive molecule, a growthfactor molecule and a therapeutic molecule as disclosed herein. Abioactive molecule further includes art conventional drugs, compounds,molecules, peptides, peptidomimetics, antibodies and fragments andmimics thereof, and the like. A bioactive molecule may, but need not,bind to a receptor in the organism, be a receptor for an endogenoussubstance found in an organism, or be an agonist, antagonist, or a mixedagonist-antagonist of a receptor-mediated process or reaction. In apreferred embodiment, the bioactive molecule may be bound to heparin byany means known in the art, including but not limited to affinitybinding.

[0047] “Growth factor molecule” refers to any growth factor, mimic of agrowth factor, derivative of a growth factor, or other molecule that hasthe effect of a growth factor. This includes fibroblast growth factor,platelet-derived growth factor, vascular endothelial growth factor,hepatocyte growth factor, placental growth factor, insulin-like growthfactor, nerve growth factors and neurotrophins, heparin-bindingepidermal growth factor, transforming growth factor-β, bonemorphogenetic protein 2 (BMP-2), osteogenic protein 1 (OP-1, also calledBMP-7) and keratinocyte growth factor. It further includes peptides,peptidomimetics and other molecules, whether made by synthetic means,recombinant means or otherwise, which have the biological activity of agrowth factor, including without limitation any of the foregoing. In apreferred, embodiment, the bioactive molecule may be bound to heparin byany means known in the art, including but not limited to affinitybinding.

[0048] “Heparin” as used herein includes complex carbohydrates ormimetics of complex carbohydrates with properties similar to those ofheparin, including heparan sulfate, hyaluronic acid, dextran, dextransulfate, chondroitin sulfate, dermatan sulfate, and the like, includingbut not limited to a molecules including a mixture of variably sulfatedpolysaccharide chains composed of repeating units of D-glucosamine andeither L-iduronic or D-glucuronic acids, salts of any of the foregoingand derivatives of any of the foregoing.

[0049] “Therapeutic molecule” refers to any molecule having atherapeutic effect, such as a chemokine, hormone, angiogenesis inhibitoror drug, and particularly a therapeutic molecule intended for local orregional delivery within the body over a sustained period. Examples ofchemokines and modulators of the immune system include C-X-C chemokines,interferon gamma, macrophage inflammatory protein-1, interleukins, suchas IL-1, IL-2, IL-3, IL-4, IL-6, IL-7 and IL-8, interferon-gammainducible protein-10, RANTES, HeIV-tat-transactivating factors, andgranulocye/macrophage-colony stimulating factor. Examples ofangiogenisis inhibitors include platelet factor-4 (PF-4), C-X-Cchemokines lacking the ELR motif (ELR-C-X-C chemokines), endostatin andangiostatin. Examples of drugs include amino glycoside antibiotics,including streptomycin, gentimicin and neomycin B; and anti-cancerantibiotics, including actinomycin D, daunorubicin, doxorubicin,bleomycin, rapamycin and paclitaxol. In a preferred embodiment, thetherapeutic molecule may be bound to heparin by any means known in theart, including but not limited to affinity binding.

[0050] In the following discussion and examples, “μL” means microliter,“mL” means milliliter; “L” means liter, “μg” means microgram, “mg” meansmilligram, “g” means gram, “mol” means moles, “M” means molarconcentration, “Me” means methyl; “Bn” means benzyl, “nBu₄NI” meanstetrabutyl-ammonium iodide, “C” means degrees Centigrade. Allpercentages are in percent by weight unless otherwise indicated.

[0051] Silyl-Heparin Compositions

[0052] The silyl-heparin compositions of the present invention include acovalent complex of one or more hydrophobic silyl moieties with heparin.Heparin is a mixture of variably sulfated polysaccharide chains composedof repeating units of D-glucosamine and either L-iduronic orD-glucuronic acids.

[0053] Any suitable form of heparin may be employed in the reaction. Avariety of salts of heparin and heparin derivatives are known in theart. For example, conventional salts of heparin include sodium heparin,calcium heparin, magnesium heparin, and potassium heparin. Heparinderivatives include, but are not limited to ammonium heparin,benzalkonium heparin, and the like. Sodium heparin is a preferred formof heparin for preparing the covalent complexes according to the presentinvention. All of the foregoing are included within the definition of“heparin” given above, together with salts and derivatives thereof

[0054] The silyl moiety is represented by Formula I wherein R₁ is anC₁₋₁₈ alkyl or C₆₋₃₂ aryl group, each R₂ is independently selected fromthe group consisting of C₁₋₁₈ alkyl and C₆₋₃₂ aryl, R₃ is N or O, and nis a number from 1 to 10. As will be apparent to those skilled in theart, R₃ is an N or O atom in the heparin molecule, and the unoccupiedbond from R₃ signifies the attachment of the silyl moiety to the heparinmolecule. Thus, the hydrophobic silyl moiety is capable of attachment tothe heparin molecule at either an 0 atom of an alcohol (i.e., hydroxyl)or an N atom of an amine.

[0055] Heparin comprises many repeating units containing amine andhydroxyl functional groups which can be the site for attachment of thehydrophobic silyl moiety to the heparin molecule. Accordingly, oneembodiment of the present invention contemplates the attachment of morethan 1 hydrophobic silyl moiety to a single heparin molecule. As many as30 or more hydrophobic silyl moieties of Formula I, and as few as 1hydrophobic silyl moiety, may be attached to a single heparin moleculeto achieve the covalent complex employed in the heparin coatingcompositions of the present invention. In one embodiment of the presentinvention, between 2 and 25 hydrophobic silyl moieties are attached to asingle heparin molecule. In another embodiment, between 5 and 20hydrophobic silyl moieties are attached to a single heparin molecule. Inanother embodiment, between 7 and 15 hydrophobic silyl moieties areattached to a single heparin molecule. In a preferred embodiment, 7 or 8hydrophobic silyl moieties are attached to a single heparin molecule. Inanother preferred embodiment 12 hydrophobic silyl moieties are attachedto a single heparin molecule.

[0056] As disclosed herein, the silyl-heparin complex is bound to thecontacting surface of a medical device by means of hydrophobicinteraction between the hydrophobic silyl moiety and the contactingsurface, which is preferably also hydrophobic. The strength of theattachment, and the disassociation rate of silyl-heparin complexes fromthe contacting surface, is determined by at least three factors: thenumber of silyl units per silyl moiety (i.e., where n is a numberbetween 1 and 10), the number of silyl moieties per single heparinmolecule (i.e., where x is a number between 1 and about 30), and thedegree of hydrophobicity of the contacting surface. For applicationswhere minimal or functionally no disassociation is desired, such ascertain applications with adhesive molecules bound to the silyl-heparincomplex, the number of silyl units per silyl moiety and the number ofsilyl moieties per heparin molecule may each be increased to the optimalnumber for maximal binding strength, and similarly the contactingsurface may be selected so as to provide optimal hydrophobic bindingwith the silyl moieties. For applications where controlled release overtime is desired, such as certain applications with growth factor ortherapeutic molecules bound to the silyl-heparin complex, fewer silylunits per silyl moiety or fewer silyl moieties per heparin molecule, orboth, are selected, or a contacting surface providing decreasedhydrophobic binding to the silyl moiety is selected, or a combinationthereof, such that the desired release over time in vivo is obtained.

[0057] In those embodiments wherein more than one hydrophobic silylmoiety is attached to a single heparin molecule, the hydrophobic silylmoieties may be attached either through the amine of heparin (e.g.,where R₃ is N) or through the hydroxyl group of heparin (e.g., whereinR₃ is O). In other words, some of they hydrophobic silyl moieties may beattached to the heparin molecule via bonding at the amine groups ofheparin, while other hydrophobic silyl moieties are attached to theheparin molecule via bonding at the hydroxyl groups of heparin. It isalso possible for all of the hydrophobic silyl moieties to beconsistently attached to heparin via one or the other of the amine(e.g., R₃ in all hydrophobic silyl moieties is N) or the alcohol (e.g.,R₃ in all hydrophobic silyl moieties is O).

[0058] The bonds between the hydrophobic silyl moieties and the heparinmolecule that effect the attachment of the silyl moieties to the heparinmolecule are covalent bonds. Thus, the coating compositions of thepresent invention do not rely upon ionic interactions between heparinand the hydrophobic moiety. Rather, the hydrophobic moieties are bondedto the heparin molecule by covalent bonding through either the amine orhydroxyl groups (or possibly a combination of both amine and hydroxylgroups when two or more hydrophobic silyl moieties are attached a singleheparin molecule). Because the hydrophobic silyl moiety is bound to theheparin molecule through covalent bonding, the present inventionovercomes one weakness of conventionally known heparin coatings.Specifically, the problem of heparin leaching from the coating as aresult of the breaking of the ionic bond between heparin and the groupwhich attaches heparin to the surface is overcome by avoiding relianceupon ionic bonding interactions between heparin and the binding group.In the present invention, the covalent bonds between the hydrophobicsilyl moieties and the heparin molecule in the coating composition arenot disrupted by the presence of ionic species in the blood with whichthe coated surface will come into contact. The data demonstrate thatthis process of covalent modification also does not lead to detrimentalloss of heparin activity as monitored by a Factor Xa/antithrombin IIIchromogenic substrate assay on the surface of target substrates.

[0059] The covalent complex according to the present invention can beprepared according to the following Scheme 1.

[0060] wherein R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group, each R₂ isindependently selected from the group consisting of C₁₋₁₈ alkyl andC₆₋₃₂ aryl, R₃ is N or O, n is a number from 1 to 10, and x is a numberfrom 1 to about 30.

[0061] Generally, the first intermediate, R₁(Si(R₂)₂CH₂)_(n)Cl, whereinn is 1, is produced by reacting an alkyl or aryl magnesium chloride witha chloro(chloromethyl)-dialkyl silane or chloro(chloromethyl)diarylsilane in the presence of tetrahydrofuran (THF). The alkyl or arylmagnesium chlorides used as starting materials are commerciallyavailable, and include, for example benzyl magnesium chloride.Chloro(chloromethyl)dialkyl silanes and chloro(chloromethyl)diarylsilanes are commercially available and include, for examplechloro(chloromethyl)dimethyl silane. The reaction is exothermic, and istypically conducted at temperatures of about 10° C. or less. Thereaction is carried out for a sufficient period of time to yield about80-90% product. Typically the reaction is conducted over a period offrom about 2 to about 24 hours.

[0062] First intermediates wherein n is 2 or higher can be producedusing a Grignard reaction involving the reaction of the firstintermediate wherein n is 1 with ClSl(R₂)₂CH₂Cl. This Grignard reactioncan be repeated any number of times to achieve the desired value for nin the first intermediate. The reaction is carried out in the presenceof a catalytic amount of iodine and THF.

[0063] The first intermediate (wherein n is 1-10) is converted to thesecond intermediate, R₁(Si(R₂)₂CH₂)_(n)OH, by reacting the firstintermediate with potassium acetate (KOAc) in dimethyl formamide (DMF),at a temperature of above about 120° C., and preferably about 135° C.,for between 12 and 24 hours. The product of this reaction is thenreacted with sodium methoxide (NaOMe) in methanol (MeOH) under refluxfor about 2 hours to achieve the second intermediate.

[0064] The second intermediate is converted to the last intermediate,R₁(Si(R₂)₂CH₂)_(n)OCO₂N(COCH₂)₂, by a two-step reaction process. In thefirst step, the second intermediate is reacted with triphosgene andsodium carbonate in methylene chloride at a temperature of less than 10°C., and preferably about 0° C. The product of this reaction is reactedwith N-hydroxysuccinimide and triethylamine (Et₃N) in methylene chlorideat a temperature of less than 10° C., and preferably about 0° C.

[0065] The final intermediate is covalently conjugated to heparin byreacting heparin with the final intermediate in a suitable solvent(e.g., water/dimethyl formamide) at a pH of about 8.0 to 9.0, andpreferably about 8.5. The pH of the reaction is controlled by theaddition of base such as sodium hydroxide, as needed. Alternatively andpreferably, a slight excess of 4-dimethylaminopyridine (DMAP) can beused as base for the conjugation reaction with heparin. Using thesegeneral methods, the covalent silyl-heparin complexes of the presentinvention can be produced. The covalent complexes have the generalFormula III:

[0066] wherein R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group, each R₂ isindependently selected from the group consisting of C₁₋₁₈ alkyl andC₆₋₃₂ aryl, R₃ is N or O of heparin, n is a number from 1 to 10, and xis a number from 1 to about 30.

[0067] Preferred complexes include those complexes wherein R₁ of thehydrophobic silyl moiety is aryl. In one preferred embodiment, R₁ isbenzyl. In one preferred embodiment, each R₂ is alkyl. In oneparticularly preferred embodiment, each R₂ is selected from the groupconsisting of methyl, ethyl, propyl, and isopropyl, particularly methyl.In one preferred embodiment, n is a number from 2 to 3.

[0068] Specific examples of covalent complexes according to the presentinvention include but are not limited to[benzyl-bis(dimethylsilylmethyl)]-(N-heparinyl)-carbamate,[benzyl-tris(dimethylsilylmethyl)]-(N-heparinyl)carbamate, anddodecyl[benzyl-bis(dimethylsilylmethyl)]-(N-heparinyl)-carbamate.Although these three specific covalent complexes are examples ofcurrently preferred covalent complexes having the general Formula IIIabove, other specific examples of such complexes will be apparent tothose skilled in the art and are contemplated by the instant invention.

[0069] The silyl-heparin coatings of the present invention include thesilyl-heparin covalent complexes described above. In addition to thesilyl-heparin covalent complex, the coating composition may also includeone or more solvents that facilitate the processes of applying thecomposition to the surface. Suitable solvents include those which atleast partially solubilize the covalent complex and which do notinterfere with the anti-thrombogenic activity of heparin. Examples ofsolvents which may be employed in the coating compositions of thepresent invention include but are not limited to aqueous solvents,alcohols, nitriles, amides, esters, ketones, ethers, and the like.“Aqueous”, with reference to solutions or solvents, refers to solutionsor solvents that consist primarily of water, normally greater than 90%water by weight, and includes essentially or substantially pure water.For example, an aqueous solution or solvent can be distilled water, tapwater, or the like. However, an aqueous solution or solvent can alsoinclude water having substances such as pH buffers, pH adjusters,organic and inorganic salts, alcohols (e.g., ethanol), sugars, aminoacids, or surfactants incorporated therein. The aqueous solution orsolvent may also be a mixture of water and minor amounts of one or moreco-solvents, including agronomically suitable organic co-solvents, whichare miscible therewith, or may form an emulsion therewith. Examples ofsuitable alcohol solvents include but are not limited to methanol,ethanol, propanol, isopropanol, hexanol, as well as glycols such asethylene glycol and the like. Examples of suitable nitriles includeacetonitrile, propionitrile, butyronitrile, benzonitrile and the like.Examples of suitable amides include formamide, N,N-dimethylformamide,N,N-dimethylacetamide and the like. Examples of suitable esters includemethyl acetate, ethyl acetate and the like. Examples of suitable ketonesinclude acetone, methyl ethyl ketone, diethyl ketone and the like.Examples of suitable ethers include diethyl ether, tetrahydrofuran,dioxane, dimethoxyethane and the like. Any two or more of any of theforegoing solvents may be utilized in the coating composition as well.Currently preferred solvents include water, particularly distilledwater, isopropanol, acetonitrile, and combinations of any two or more ofthese solvents.

[0070] In one preferred embodiment, the silyl-heparin covalent complexis solubilized in solvent to achieve a concentration of between about0.01 and about 10 percent by weight, preferably between about 0.1 andabout 1 percent, and more preferably about 0.125 percent.

[0071] In addition to the foregoing solvents, the silyl-heparin coatingcompositions of the present invention may also include therein variousconventional additives. Examples of additives which may be incorporatedinto the compositions of the present invention include but are notlimited to benzalkonium, 4-dimethylaminopyridinium, tetrabutylammoniumhalides, and the like.

[0072] Contacting Surfaces of Medical Devices

[0073] The coatings may be applied to any of a wide variety ofcontacting surfaces of medical devices. Contacting surfaces include, butare not limited to, surfaces that are intended to contact blood, cellsor other bodily fluids or tissues of a mammal, including specifically ahuman. Suitable contacting surfaces include one or more surfaces ofmedical devices that are intended to contact blood or other tissues. Themedical devices include sutures, graft materials, wound dressings, woundcoverings, bone waxes, bone prostheses, aneurysm coils, embolizationparticles, microbeads, stents, catheters, shunts, grafts, artificialblood vessels, nerve-growth guides, artificial heart valves,prosthetics, pacemaker leads, in-dwelling catheters, cardiovasculargrafts, bone replacement, wound healing devices, cartilage replacementdevices, urinary tract replacements, orthopedic implants, opthalmicimplants and other medical devices known in the art. Other examples ofmedical devices that would benefit from the application of the presentinvention will be readily apparent to those skilled in the art ofsurgical and medical procedures and are therefore contemplated by theinstant invention. The contacting surface may include a mesh, coil,wire, inflatable balloon, or any other structure which is capable ofbeing implanted at a target location, including intravascular locations,intralumenal locations, locations within solid tissue, and the like. Theimplantable device can be intended for permanent or temporaryimplantation. Such devices may be delivered by or incorporated intointravascular and other medical catheters.

[0074] Suitable contacting surfaces include metals such as stainlesssteel, nitinol, titanium, tungsten, platinum, graphite and metal alloys;ceramics; any of a variety of polymeric materials such as polyvinylchloride, polyethylene, polylactide, polyglycolide, polycaprolactone,polymethyl methacrylate, polyhydroxylethyl methacrylate, polyurethane,polystyrene, polycarbonate, dacron, polytetrafluoroethylene and extendedpolytetrafluoroethylene (Teflon®), related fluoropolymer composites(Gore-Tex®), polyester, polypropylene, polyamide, polyacrylate polyvinylalcohol and copolymers of any two or more of the foregoing; siloxanessuch as 2,4,6,8-tetramethylcyclotetrasiloxane; natural and artificialrubbers; and glass. In general, the contacting surface that may becoated with a silyl-heparin-bioactive molecule of the present inventionincludes any surface that has an affinity or attraction to thehydrophobic silyl moiety. Such surfaces are typically hydrophobicsurfaces.

[0075] In one embodiment, the contacting surface may be a biodegradableor bioerodible material. Biodegradable or bioerodible materials areknown in the art, and include polyanhydrides, polyglycolic acid,polylactic/polyglycolic acid copolymers, polyhydroxybutyrate-valerateand other aliphatic polyesters. Biodegradable implantable materials aredescribed in U.S. Pat. Nos. 5,656,297; 5,543,158; 5,484,584; 4,897,268;4,883,666; 4,832,686; and 3,976,071. In one embodiment, a bioabsorbablepolymeric contacting surface is made from a biocompatible polymericmaterial such as polycaprolactone, poly(D,L-lactide), poly(L-lactide),polyglycolide, poly(dioxanone), poly(glycolide-co-trimethylenecarbonate), poly(L-lactide-co-glycolide),poly(D,L-lactide-co-glycolide), poly(L-lactide-co-D,L-lactide) orpoly(glycolide-co-trimethylene carbonate-co-dioxanone). In oneembodiment, the persistence of the bioabsorbable polymeric structuralcomponent within a living organism is in excess of the anticipatedperiod over which the bioactive agent will provide a therapeutic effect,and is preferably in excess of at least two such periods.

[0076] Application of Silyl-Heparin Complex

[0077] The silyl-heparin complex may be coated onto any of thecontacting surfaces as set forth above. Any suitable method for applyingthe silyl-heparin complex to the surface may be employed. One suitablemethod for applying the silyl-heparin complex to the contacting surfaceis by dipping the contacting surface into a coating compositioncontaining the silyl-heparin complex of the present invention. A liquidcoating composition containing the silyl-heparin complex of the presentinvention may be prepared using any of the solvents described above. Thesurface is dipped or immersed into a bath of the coating composition.Typically, the dipping process is carried out at elevated temperatures,such as between about 30° C. and about 80° C., and for a defined periodof time, such as for a period of between about 5 and about 20 minutes,preferably about 10 minutes. Thereafter, the surface may be allowed toremain in contact with the coating composition containing thesilyl-heparin complex for a period of between about 15-60 minutes,preferably about 20 minutes, at room temperature.

[0078] Another method that may be employed for coating or applying thecoating compositions of the present invention onto a contacting surfaceincludes use of pumping or spraying processes. In a pumping process, acoating composition having a concentration of between 0.05 and about 5percent (w/v) silyl-heparin complex is pumped through that portion of amedical device including a contacting surface for about 30 minutes.Thereafter any excess coating composition may be washed out with wateror saline. Similarly, the contacting surface may be coated with thecoating composition by spraying.

[0079] Following coating, the resulting silyl-heparin complex coatedsurface is typically washed with water or other aqueous solutions priorto drying. Advantageously, the foregoing methods for applying thecoating composition to a surface are relatively quick, commerciallyfeasible and cost-effective. They require no special equipment orspecial technical training, and can be applied to devices with complexsurface geometries. Thus the silyl-heparin complex may be applied to anycontacting surface, including three-dimensional matrices, rods, coils,tubes, sheets, pins, screws, threads, braids, beads, particles, spheresand combinations thereof.

[0080] The hydrophobic interaction between the hydrophobic contactingsurface and the hydrophobic silyl moieties of the silyl-heparin complexforms a bond between the silyl moiety of the silyl-heparin complex andthe surface. This hydrophobic interaction is sufficiently strong so asto provide a reasonable stable bond between the covalent complex and thesurface. Advantageously, and depending in part on the hydrophobicity ofthe material of the contacting surface, a definable disassociation ratemay be obtained, such that a given percentage of silyl-heparincomplexes, together with the bioactive molecule bound to heparin,disassociate from the contacting surface, thereby providing for local orregional delivery of the bioactive molecule over a period of time. Suchdisassociation rate may be determined, in part, by the number of silylunits per silyl moiety or the number of silyl moieties bound to eachheparin molecule, or both, and accordingly such numbers are selectedbased, in part, on the desired disassociation rate.

[0081] Adhesive Molecule Complexation and Use

[0082] The bioactive molecule may be an adhesive molecule such asfibronectin. Fibronectin has known and demonstrated affinity forheparin. Fibronectins are composed of two similar protein chains, witheach chain including one domain for cell binding and two domains, one ateach end of the chain, for heparin binding. Affinity binding offibronectin results in an affinity constant of approximately 10⁸ M/L.

[0083] Any form of fibronectin may be employed, including fibronectinderived from cells, plasma, or tissues, natural or geneticallyengineered, and of human origin or derived from another animal species.Other adhesive molecules, as defined above, may also be employed,utilizing the methods described herein for fibronectin.

[0084] Following coating of the contacting surface with a silyl-heparincomplex, the fibronectin may be affinity complexed to the heparin,resulting in a silyl-heparin-fibronectin complex. To affinity complexthe fibronectin, the fibronectin is solubilized in one or more solventswhich facilitate the processes of applying the fibronectin compositionto the silyl-heparin-coated contacting surface. Suitable solventsinclude those which at least partially solubilize fibronectin and whichdo not interfere with the activity of heparin or the cellular-attachmentactivity of fibronectin. Examples of solvents that may be employed inthe present invention include aqueous solutions as defined above;aqueous solutions containing alcohols, nitriles, amides, esters,ketones, ethers, and the like; and alcohols, nitrites, amides, esters,ketones, ethers, and the like. Aqueous solutions are particularly usefulfor non-synthetic fibronectins and organic-based solvents areparticularly useful for synthetic fibronectins and peptides derivedtherefrom.

[0085] The fibronectin composition can be applied to thesilyl-heparin-coated contacting surface to render the contacting surfacesuitable for cellular adhesion and attachment. Any suitable method forapplying the fibronectin composition to the surface may be employed. Onesuitable method for applying the fibronectin composition to thesilyl-heparin-coated contacting surface is by dipping thesilyl-heparin-coated contacting surface into a fibronectin compositionof the present invention. The silyl-heparin-coated surface is dipped orimmersed into a bath of the fibronectin solution. Typically, the dippingprocess is carried out at elevated temperatures, such as between about30° C. and about 80° C., and preferably between about 40° C. and about50° C., for a period of between about 5 and about 60 minutes, preferablybetween about 20 and about 30 minutes.

[0086] Other methods that may be employed for coating or applying thefibronectin solutions of the present invention on tosilyl-heparin-coated surfaces include use of pumping or sprayingprocesses. In one embodiment, the fibronectin solution has aconcentration of between 0.05 and about 5 percent (w/v) fibronectin, andis pumped through that portion of a medical device including acontacting surface for about 30 minutes. Thereafter any excess coatingcomposition may be washed out with water or saline. Similarly, thecontacting surface may be coated with the coating composition byspraying.

[0087] Following attachment of the fibronectin to the heparin of thesilyl-heparin complex of the composition onto the contacting surface,the contacting surface may be washed with water or saline prior todrying. Advantageously, the foregoing methods for applying the coatingcomposition to a surface are relatively quick, commercially feasible andcost-effective.

[0088] In an alternative embodiment, the fibronectin may be complexed inan aqueous solution with silyl-heparin, and then the entiresilyl-heparin-fibronectin complex attached to the contacting surface. Inthis embodiment, the silyl-heparin is dissolved in an aqueous solutionand mixed with a predetermined amount of fibronectin, such that thefibronectin is essentially entirely complexed with the silyl-heparin. Anorganic solvent such as isopropanol may be added to the aqueous solutionto a concentration of between 20% and 80%, and preferably about 35%.Under these conditions the silyl-heparin-fibronectin complex undergoesthe formation of micelles, with the fibronectin-heparin portion of thecomplex sequestered on the inside of the micelle. A solution of micellescan then be applied to the surface of a contacting surface, therebyallowing the micelles to associate with such contacting surface byhydrophobic interaction. Rinsing in an aqueous solution may then beemployed to remove excess unbound material and allow theheparin-fibronectin to associate in the aqueous phase. This approach mayadvantageously be used with, for example, fibronectin-derived peptidepolymers.

[0089] In yet another embodiment, other adhesive molecules may beemployed in addition to the adhesive molecules discussed above. Theseinclude, without limitation, polylysine, polyornithine, and similarmolecules with net positive charges that associate with heparin bycharge-charge interaction and thereby provide cell adhesive properties.Polylysine and polyornithine are known to enhance cell attachment oncertain types of cell culture ware, and may be applied and bound tosilyl-heparin complexes as described herein.

[0090] A variety of growth factors and cytokines can also be complexedto extracellular matrix heparin or heparan sulfate and employed asadhesive molecules. The bound growth factors can thereby be used topromote cell adhesion by providing a display of ligands to which cellsurface receptors can bind. Fibroblast growth factor (FGF),platelet-derived growth factor (PDGF), and bone morphogenic protein(BMP) are examples of growth factors that complex to heparin. Similarly,cytokines are known to interact with heparin, and cytokines, such asgamma-interferon, may be bound to extracellular matrix heparin orheparan sulfate compositions of this invention, including thesilyl-heparin complexes.

[0091] In yet another embodiment, more than one type of adhesivemolecule may be bound to silyl-heparin complex. Thus, both a growthfactor and fibronectin may be applied to the silyl-heparin complex suchthat the composition contains both types of adhesive molecules. For manyadhesive molecules, affinity binding to heparin is through distinctreceptors, such as with fibronectin and a fibroblast growth factor, forexample. The bound growth factor thereby provides a mechanism tomaximize cellular growth on the coated surface.

[0092] The heparin and fibronectin coating compositions of the presentinvention can be applied to contacting surfaces that contact blood,tissue, cells or other bodily constituents of any of a wide variety ofmedical devices, thereby providing the medical device with one or morecontacting surfaces promoting cellular adhesion and attachment. Examplesof specific medical devices which may be advantageously treated with thea silyl-heparin-adhesive molecule complex include, but are not limitedto, artificial blood vessels, blood shunts, nerve-growth guides,artificial heart valves, prosthetics, pacemaker leads, in-dwellingcatheters, cardiovascular grafts, bone replacements, wound healingdevices, cartilage replacement devices, urinary tract replacements andthe like. Other examples of medical devices which would benefit from theapplication of the adhesive molecule coating compositions of the presentinvention will be readily apparent to those skilled in the art ofsurgical and medical procedures and are therefore contemplated by theinstant invention.

[0093] It is also possible and contemplated to use these coatings inother applications, include use for cell culture and other biological exvivo applications wherein a surface for cellular adhesion is desired.

[0094] Growth Factor Molecule Attachment and Use

[0095] Heparin-containing delivery systems for local or regionalapplication of growth factors have been described by severalinvestigators. When the heparin is covalently bound to a scaffolding,the release of growth factors is understood to be related to enzymaticfactors such as heparinase or plasmin, and more significantly bydiffusion-based release of the growth factor from the heparin. Diffusionis, however, not favored because the complex of growth factor andheparin is stable and is rendered immobilize by the conjugation ofheparin. In this invention, the silyl-heparin complex is not covalentlybound to the contacting surface, but is rather bound by hydrophobicinteraction. As a consequence, the complex of silyl-heparin-growthfactor molecule is subject to efflux and more effective biologicaleffect.

[0096] A growth factor molecule may be adsorbed onto the heparin surfaceof a silyl-heparin complex bound by hydrophobic interaction to acontacting surface of a medical device. Such adsorption may be bydipping, pumping, spraying or other means known in the art, includingthose discussed above with respect to adhesive molecules.

[0097] Silyl-heparin-growth factor molecules complexes were attached tosuitable contacting surfaces, including polystyrene andlactide:glycolide copolymers. Immunoassays were used to establish thatbasic fibroblast growth factor (bFGF) readily bound to silyl-heparincomplexes on a contacting surface, and that the amount of bFGF bound wasdirectly related to amount offered for binding. Once adsorbed thesilyl-heparin-bFGF was able to induce capillary tube formation ofendothelial cells and to increase the growth of endothelial cells. Whencoated onto suture material as a contacting surface and implanted inmuscle, the silyl-heparin-bFGF coating caused increased fibroblasticcellularity in the area of the implant. The devices and methods of thisinvention may thus be employed in a growth factor delivery system foruse in tissue repair providing localized release of bFGF and relatedgrowth factors to enhance the repair process.

[0098] Fibroblast growth factors comprise a large family ofdevelopmental and physiological signaling molecules. All FGFs have ahigh affinity for the glycosaminoglycan heparin and for cell surfaceheparan sulfate (HS) proteoglycans. The affinity of FGF for heparansulfate limits its diffusion and restricts its release into theextracellular matrix. The binding of FGFs to heparin or HS results inthe formation or stabilization of dimmers and higher order oliomersalong the proteoglycan chain. Heparin and HS also play a role in theformation of an active FGF/FGF receptor signaling complex, and heparinand HS increase the affinity and half-life of the resulting FGF andreceptor complex. bFGF is mitogenic in many cell types includingfibroblasts, endothelial cells, smooth muscle cells and osteoblasts,among others. It is angiogenic and a survival factor.

[0099] Examples of medical devices with which silyl-heparin-growthfactor molecule complexes may be employed include sutures, graftmaterials, wound dressings, wound coverings, nerve growth guides, bonewaxes, aneurysm coils, embolization particles, microbeads, stents,dental implants, orthopedic implants, optholmic implants and boneprosthesis. A single silyl-heparin-growth factor molecule complex may beutilized, two or more silyl-heparin-growth factor molecule complexes maybe utilized, or a silyl-heparin complex may include a growth factormolecule in combination with either an adhesive molecule or therapeuticmolecule, or both. Similarly, in a single medical device more than onecontacting surface may be employed, such that one contacting surfacecontains one or more silyl-heparin-growth factor molecule complexes, andanother contacting surface contains a different silyl-heparin-growthfactor molecule complex, or a silyl-heparin-adhesive molecule complex,or a silyl-heparin-therapeutic molecule complex, or a combinationthereof. For example, a medical device may have one contacting surfacethat contacts tissue, with a silyl-heparin-adhesive molecule complexcoating thereon, and another contacting surface that contacts blood,with a silyl-heparin-growth factor molecule complex or asilyl-heparin-therapeutic molecule complex coating thereon.

[0100] Disease states wherein silyl-heparin-growth factor moleculecomplex coatings on coating surfaces may be of particular utilityinclude burns, cardiovascular ischemia, peripheral vascular ischemia,vascular aneurysms, bone fractures, skeletal defects, sites oforthopedic trauma, sites of cartilage damage, cancer treatment,prophylaxis of bacterial growth, neural damage, myocardial infarction,peripheral vascular occlusion, ocular degeneration, kidney ischemia andthe like.

[0101] In one embodiment, a silyl-heparin-growth factor molecule complexcoating is employed for use in treatment of cerebral aneurysms.Endovascular coils, such as those made from platinum, titanium, ornitinol, with a diameter of about 0.015 cm to 0.031 cm and lengths of 4cm to 10 cm are employed. The surface of the endovascular coil is coatedwith a silyl-heparin-growth factor molecule complex, such asbenzyl-bis(dimethylsilylmethyl) oxycarbamoyl-heparin bound byhydrophobic interaction to the contacting surface of the endovascularcoil, with bFGF bound by affinity binding to the heparin. The coatedendovascular coil is then deployed, such as from a catheter guided underfluorography to the aneurysm. After deployment within the cavity of theaneurysm, the coil is detached from the delivery device. The number ofsilyl moieties attached to each heparin molecule or the number of silylunits in each silyl moiety, or both, is selected such as to providedisassociation of the silyl-heparin-growth factor molecule complex fromthe coil at a determined and desired rate. This provides an improvedendovascular coil with a mechanism to stimulate repair and strengtheningof the aneurysm vessel wall. The mechanism involves, in part, theheparin and bFGF stimulating transient and local cell growth leading toa stronger blood vessel wall, such effect being modulated and increasedby release of the silyl-heparin-bFGF complex from the endovascular coilat a determined rate.

[0102] It has been determined by use of immunoassays that bothfibrinogen and bFGF can be bound to heparin on a silyl-heparin complex,without displacement of either, resulting in formation of a mixedcomplex of silyl-heparin and fibrinogen and bFGF. Such mixed complexesmay be employed to coat endovascular coils and contacting surfaces ofother medical devices. In the case of endovascular coils, thesilyl-heparin-fibrinogen contributes to efficacy of thrombus formationand cellular attachment due to effect of the fibrinogen, while thesilyl-heparin-bFGF contributes to cell growth.

[0103] In another embodiment, a silyl-heparin-growth factor complexcoating is used on microparticles for local and transient delivery ofheparin and growth factor to increase collateral blood flow and toincrease angiogenesis in ischemic tissue. Microparticles are made, suchas by homogenizing a methylene chloride solution containing 3%polylactide:polyglycolide (PLGA) with a 2% polyvinyl alcohol aqueoussolution to form an emulsion, sonicating the resulting mixture, andrecovering particles by centrifugation after evaporation of the organicphase. After evaporation of methylene chloride, the PLGA microparticlesare incubated in an aqueous solution containing 0.5% silyl-heparin, suchas benzyl-bis(dimethylsilylmethyl) oxycarbamoyl-heparin, for 45 minutes.Unbound silyl-heparin is removed by centrifugation and rinsing inphosphate buffered saline. The silyl-heparin coated microparticles canthen be resuspended in an aqueous buffer, such as a buffer containingdextrose, and optionally aliquoted into unit doses, lyophilized, andoptionally sterilized by gamma radiation. The growth factor molecule canthen be incorporated immediately prior to use, by preparing growthfactor molecules in an aqueous solution, such as phosphate bufferedsaline containing 1% human serum albumin, and added the growth factormolecules and solution to the lyophilized silyl-heparin coatedmicroparticles. Alternatively, the microparticles are coated with asilyl-heparin-growth factor complex, such as by coating withsilyl-heparin and subsequently adding the growth factor molecule,incubating the microparticles with growth factor molecules at thedesired concentration, removing unbound growth factor by centrifugationand rinsing. The silyl-heparin-growth factor molecule complex coatedmicroparticles are then optionally lyophilized and sterilized asdescribed above. Any of a variety of growth factors may be employed,including bFGF, transforming growth factor-β (TGF-β) and vascularendothelial growth factor (VEGF). The silyl-heparin-growth factor isreleased from the microparticles over a selected period of time, such asseveral days, thereby providing local and transient delivery of heparinand growth factors. In one embodiment, the length of the silyl moietymay be modified, by appropriately selecting “n” in Formula I or II andaccordingly synthesizing the resulting silyl moiety, thereby modifyingthe rate of release or disassociation from the contacting surface of themicroparticle, and thus controlling bioavailability in vivo. Similarly,the number of silyl moieties per heparin molecule may be modified, byappropriately selecting “x” in Formula I or II, thereby also modifyingthe rate of release or disassociation from the contacting surface of themicroparticle, and thus controlling bioavailability in vivo.

[0104] Therapeutic Molecule Attachment and Use

[0105] Therapeutic molecules have been used in a wide variety of drugdelivery systems, methods and devices, well known in the art, for localor regional delivery of therapeutic molecules. The methods describedabove with respect to adhesive molecules and growth factor molecules maybe employed with therapeutic molecules. In a preferred embodiment, atherapeutic molecule is selected which binds, by affinity binding orother means, to heparin, thereby resulting in formation of asilyl-heparin-therapeutic molecule complex.

[0106] Industrial Applicability

[0107] The invention is further illustrated by the followingnon-limiting examples.

EXAMPLE 1

[0108] Method for Preparing Silyl-Heparin Covalent Complexes.

[0109] Treatment of benzylmagnesium chloride 1 withchloro(chloromethyl)dimethysilane 2 gavebenzyl(chloromethyl)dimethysilane 3 (n=1), all as shown in Scheme 2below. Treatment of 3_(n) with magnesium gave the Grignard Reagent,which was treated with chloro(chloromethyl)dimethyl-silane again to givethe homologous silyl compound 3_(n+1). The Grignard reaction can berepeated over and over again to obtain the desired chain-length for thesilyl compound. The chloro-silyl compound 3, on treatment with potassiumacetate, followed by trans-esterification of the corresponding acetatewith basified methanol gave the alcohol 4. The alcohol 4, when treatedwith triphosgene gave the corresponding chloroformate, which ontreatment with N-hydroxy-succinimide gave theN-hydroxy-succinimidyl-carbonate 5. The conjugation of heparin with 5was achieved by treatment of heparin with 5 and4-(dimethylamino)pyridine in 1:1 DMF/H₂O to give the silylated heparin6. Adjusting the molar ratios of the reactants controlled the number ofprosthetic groups per heparin.

[0110] Synthesis of Benzyl(chloromethyl)dimethylsilane, 3 (n=1). Underan atmosphere of nitrogen, chloro(chloromethyl)dimethylsilane (100 mL,0.744 mol) was added by syringe to THF (500 mL) and solution cooled to0° C. with an ice/acetone bath. Benzylmagnesium chloride (2.0 Msolution, 400 mL, 0.8 mol) was added dropwise over 2 hours. Care wastaken to maintain the temperature below 100 C until all the reagent wasadded. Thereafter, the ice bath was allowed to warm up to roomtemperature and the reaction mixture stirred overnight. Hexane (300 mL)was added and saturated aqueous NH₄Cl (300 mL) added dropwise. Thereaction mixture was transferred to a 2 L separatory funnel with morehexane (300 mL). After partitioning, the organic layer was washed withsaturated aqueous NH₄Cl (200 mL) and saturated aqueous NaCl (200 mL).The combined aqueous layers were backwashed with hexane (2×500 mL) andthe combined organic layers dried over MgSO₄, evaporated on a rotaryevaporator, and further concentrated under vacuum to give a colorlessoil 162.0 g (109.5% yield). A quantitative yield was assumed with apurity of the crude product being 91.3%.

[0111] Grignard Reaction of Bn(SiMe₂CH₂)_(n)Cl3_(n) and ClSiMe₂CH₂Cl togive Bn(SiMe₂CH₂)_(n+1)Cl, 3_(n+1). Magnesium powder (7.5 g, 0.31 mol),a catalytic amount of iodine and THF (100 mL) were placed in a 500 mL3-necked flask equipped with a condenser-N₂ inlet, a septum and athermometer. The mixture was heated to reflux briefly until the browncolor of iodine disappeared. Bn(SiMe₂CH₂)_(n)Cl (0.2 mol) was added bysyringe and residual reagent washed into the reaction mixture with THF(2×25 mL). The reaction was initiated with a heat gun: an exothermicreaction was observed and the reaction flask was placed in a water bathuntil the exothermic reaction subsided. The resulting gray mixture washeated to reflux for 24 hours. The reagent was then cooled to roomtemperature and cannulated into a pressure filter funnel where it wasadded directly into another 500 mL round bottom flask in which wasplaced a solution of ClSiMe₂CH₂Cl (27.0 mL, 0.2 mol) in THF (50 mL) atroom temperature. The magnesium residue was washed into the reactionmixture with THF (2×25 mL). The reaction mixture was heated to refluxovernight. To the resulting gray suspension was added saturated aqueousNaHCO₃ (50 mL) and the resulting solution transferred to a 500 mLseparatory funnel with hexane (200 mL). After partition, the organiclayer was washed with saturated aqueous NaHCO₃ (50 mL) and saturatedaqueous NaCl (50 mL). The combined aqueous layers were back-washed withhexane (2×100 mL), dried over MgSO₄, and extensively evaporated undervacuum to give an amber oil. The amber oil was purified by distillationto give colorless oil. Yields were over 80%.

[0112] Conversion of Bn(SiMe₂CH₂)_(n)Cl 3_(n) to Bn(SiMe₂CH₂)_(n)OH4_(n). Bn(SiMe₂CH₂)_(n)Cl (0.16 mol) was dissolved in DMF (300 mL) in a1-L 3-necked flask. KOAc (50 g, 0.5 mol) was added followed by nBu₄Nl(4.0 g, 0.01 mol) and the reaction mixture was stirred in a 135° C. oilbath for 24 hours. The reaction mixture was worked up by cooling to roomtemperature and transferring to a 1 L separatory funnel with hexane (500mL) and washed with saturated aqueous NaCl (3×100 mL). The combinedaqueous layers was back-washed with hexane (3×300 mL) and the combinedorganic layers dried over MgSO₄ and vacuum-evaporated to an amber oil,which was dissolved in MeOH (400 mL). A generous amount of freshlyprepared NaOMe was added to adjust the pH to >10 and the reactionmixture heated to reflux for 2 hours. The reaction mixture wasneutralized with AcOH, evaporated to dryness and chromatographed withsilica gel in a 6.5×100 cm (height of silica 40 cm) flash column andeluted with 0-30% EtOAc/hexane to give the desired product, a slightlyyellow oil. Yields were over 80%.

[0113] Conversion of Bn(SiMe₂CH₂)_(n)OH 4_(n) toBn(SiMe₂CH₂)_(n−)OCO₂N(COCH₂)₂ 5_(n). Triphosgene (60 g, 0.2 mol) wasdissolved in CH₂Cl₂ (200 mL) and stirred at 0° C. under N₂ in a 1-L3-necked flask equipped with thermometer, dropping funnel and N₂ inlet.Na₂CO₃ (65 g, 0.6 mol) was added followed by Bn(SiMe₂CH₂)_(n)OH (0.13mol dissolved in 200 mL of CH₂Cl₂) dropwise over 30 minutes. Thereafter,the ice/acetone bath was allowed to come to room temperature. Thereaction mixture was stirred overnight, filtered through a sinteredglass funnel, and the reaction vessel rinsed with PhCH₃ (200 mL). Thesolution was concentrated under vacuum to give a colorless oil. The oilwas dissolved in CH₂Cl₂ and stirred in an ice bath under N₂.N-Hydroxysuccinimide (30 g, 0.26 mol) was added followed by Et₃N (40 mL,0.28 mol) dropwise over 15 minutes and the resulting cloudy mixturestirred at room temperature for one hour. The reaction mixture wasdiluted with hexane (600 mL), washed with saturated aqueous NH₄Cl (3×100mL), and the combined aqueous phases backwashed with hexane (2×200 mL).The combined organic phases were dried over MgSO₄ and concentrated undervacuum to give amber oil. The amber oil was chromatographed on silicagel in a 6.5×100 cm (height of silica 40 cm) flash column and elutedwith 20-50% EtOAc/hexane to give an amber syrup. Yields are generallygreater than 75%.

[0114] Conjugation of Heparin with Bn(SiMe₂CH₂)_(n)OCO₂N(COCH₂)₂ 5_(n)to give 6_(n,x). Heparin (ammonium ion-free, average molecular weight10,000; 100 g, 10 mmol) was dissolved in H₂O (500 mL) in a 2 L flaskwith stirring. DMF (400 mL) was added followed by DMAP (1.6×g, 13×mmol).Bn(SiMe₂CH₂)_(n)O—CO₂N(COCH₂)₂, 5_(n) (10×mmol) in DMF (100 mL) wasadded and the resulting milky mixture was allowed to stir at roomtemperature for >24 hrs. The reaction mixture was concentrated byevaporating most of the water followed by trituration with acetone (2L). The white suspension was filtered through a sintered glass funnel togive a white solid residue. This crude material was purified by soxhletextraction with acetone overnight to give a white powder. The yieldswere generally greater than 95%.

[0115] The average number of prosthetic units per molecule of heparinwas estimated based on a comparison of the molar ratios of thehydrolyzed prosthetic unit benzyl-tri(dimethylsilyl-methyl)-OH andheparin as determined by use of dimethyl methylene blue. Heparin wasdetected using a commercially-available enzyme-linked assay thatmeasures the heparin-induced inhibition of antithrombin/factor Xa asmeasured with a factor Xa specific chromogenic substrate.

EXAMPLE 2

[0116] Techniques for Applying Silyl-Heparin Coating Compositions toSurfaces.

[0117] The silyl-heparin complex of Example 1 was used as a coatingsolution as a 1% solution (w/v) in 60% aqueous ethanol. Materials werecoated with silyl-heparin for 15 minutes at 37° C. The coated materialswere then were rinsed extensively in water, air-dried, and stored untiluse.

[0118] In another embodiment, the silyl-heparin complex was dissolved indistilled water with gentle stirring, and an organic solvent added, suchas isopropyl alcohol or acetonitrile, such that the organic solventconstituted approximately 2/3 of the volume, such as 100 mg ofsilyl-heparin complex of Example 1 solubilized in 27 mL of distilledwater with gentle stirring, with 53 mL of isopropyl alcohol oracetonitrile then added, providing a resulting concentration of thesilyl-heparin complex in solution of about 0.125%. In other embodiments,the resulting solution had a silyl-heparin complex concentration ofbetween 0.01 and 10 percent based upon the weight of the solution. Thematerial to be coated was dipped in the solution at elevatedtemperatures usually ranging from 30° C. to 50° C. for about 10 minutes,followed by standing in room temperature for about 20 minutes. Thecoated material was taken out of the coating solution and rinsedthoroughly with distilled water or saline solution prior to drying.

EXAMPLE 3

[0119] Stability of Heparin Coating Compositions on Surfaces Exposed toIonic Environments.

[0120] Various surfaces coated according to Example 2 were evaluated forheparin activity after washing with 3% (by weight) sodium chloridesolution. Surface heparin activity was measured in mlU/cm² according tothe technique described in Sigma Diagnostics, Heparin, Procedure No. CRS106.

[0121] Results obtained from the evaluation of average heparin activityon various surfaces after washing with sodium chloride are set forth inTable 1 below. The concentration of the covalent complex in the coatingsolution was 0.25% (W/V). TABLE 1 Percent by volume of isopropyl alcoholin IPA/H₂0 solvent 50% 55% 60% 65% 70% 75% Material Coated mIU ofHeparin/Square Centimeter POLYCARBONATE 6.8 8.0 17.6 16.3 14.7 14.9TMCTS 0.4 3.8 3.5 2.0 1.1 2.4 POLYESTER 4.5 4.4 5.5 3.7 5.3 POLYVINYLCHLORIDE 2.6 2.9 10.0 6.5 4.0 2.8 STAINLESS STEEL 12.9 13.1 8.3 11.012.9 13.8

EXAMPLE 4

[0122] Silyl-Heparin Application Prior to Fibronectin Attachment.

[0123] Benzyl magnesium chloride was treated serially withchloro(chloromethyl) dimethylsilane to give a benzyl-(1,2tetramethyl)disilyl compound. The benzyldisilyl compound was modified toform an activated N-hydroxy-succinimidyl carbonate that was, in turn,conjugated to heparin to form a benzyl-(1,2 dimethyl)disilyl heparin.This is shown in Scheme 3 below.

[0124] The silyl-heparin was used as a 1% solution in 70% acidified,aqueous ethanol. To coat wells, the silyl-heparin solution was appliedin 20 μL/well for 15 minutes at 50-60° C. The wells were then rinsedseveral times in saline and air-dried. To coat contacting surfaces, thecontacting surface was immersed in a 1% silyl-heparin solution for 15minutes at 50-60° C., and rinsed extensively in water or saline.

[0125] The silyl-heparin complex may be applied to any polymericsubstrate, either forming a medical or other implantable device, orcoated or otherwise forming a surface of a medical or other implantabledevice. The complex was applied, as described, to polystyrene andpolyurethane polymeric surfaces. The polymeric substrate includesbiodegradable polymers, including, for example, polylactide,polylactide:polyglycolide and polycaprolactone, to which thesilyl-heparin complex was applied. The silyl-heparin complex may also beapplied to any metallic substrate.

EXAMPLE 5

[0126] Attachment of Fibronectin to Silyl-Heparin Coated Substrate.

[0127] Fibronectin was attached to silyl-heparin coated contactingsurfaces of Example 4 by incubation of in an aqueous 0.9% salinesolution and 20 μg/mL bovine plasma fibronectin. After 30 minutes theunbound fibronectin was removed by rinsing. Thesilyl-heparin-fibronectin coated contacting surfaces were then eitherused directly or air dried and stored for subsequent use.

EXAMPLE 6

[0128] Detection of Heparin and Fibronectin.

[0129] Using contacting surfaces coated with silyl-heparin as in Example4, and to which silyl-heparin-fibronectin was attached as in Example 5,the presence of both heparin and fibronectin was detected in analyticalassays. Heparin was detected using a commercially-available anenzyme-linked assay kit (Sigma Chemical Co., St. Louis) that measuredthe heparin-induced inhibition of antithrombin/factor Xa as measuredwith a factor Xa specific chromogenic substrate. The enzyme-inhibitionassay was performed in low-attachment 96 well plates following thedirections of the manufacturer.

[0130] To insure that the amphipathic heparin did not introduce anunknown variable in the enzyme inhibition assay, a second assay wasperformed using a hydrazine-activated biotin in low-attachment 96 wellplates. A solution of hydrazine-activated biotin was added to wells thatwere uncoated or coated with silyl-heparin complex. The solution wascomposed of 0.1 M sodium acetate, pH 5.2, containing 350 g of EZ-linkbiotin-LC-hydrazine (Pierce Chemical Co.). After 30 minutes, the wellswere rinsed in water and quenched in 5% dextrose. A solution of PBScontaining 20% serum and a 1:1000 dilution of horseradishperoxidase-conjugated avidin (HRPO-avidin) was added. After 30 minutesthe unbound material was rinsed and a chromogenic solution of chromogen(2,2′-azinobis 3-ethylbenzothiazoline-6-sulfinic acid) (1-Step ABTS,Pierce Chemical Co.) added. Upon color development, an aliquot of 0.2 Msulfuric acid was added to stop the reaction, and the absorbancedetected at 650 nm.

[0131] Fibronectin was detected immunochemically. The assays wereperformed in low-attachment 96 well plates in wells with no coating, acoating of fibronectin, or a coating of silyl-heparin/fibronectin. PBScontaining a saturating amount of gelatin and rabbit anti-humanfibronectin (known to be cross-reactive with bovine fibronectin) wasadded to the wells. After 30 minutes the primary antibody was removed,the plate rinsed, and a solution of PBS containing a saturating amountof gelatin and goat anti-rabbit IgG was added. After 30 minutes theunbound material was rinsed off and a chromogenic solution of ABTS(1-Step ABTS, Pierce Chemical Co.) added. Upon color development, analiquot of 0.2 M sulfuric acid was added to stop the reaction, and theabsorbance detected at 650 nm.

EXAMPLE 7

[0132] Cells Used for Adhesion Studies.

[0133] Several cell types were used, including GS-9L cells (ratgliosarcoma), C3H10T1/2 (murine fibroblasts), Jurkat (human T cell),bovine aorta endothelial (BAE) cells and rat lymphocytes. GS-9L andC3H10T1/2 cells were maintained in log phase growth and detached fromcultureware using Versene and collected by centrifugation. Jurkat cellsgrew as single cell suspensions and were collected by centrifugation.Rat lymphocytes were collected from heparinized blood by densitygradient isolation over Ficoll-Hypaque® media. The collected cells wererinsed once by low speed centrifugation and suspended in eitherserum-free Dulbecco's Modified Eagles Medium (DMEM) containing pyruvateor RPMI 1640 containing 10% fetal bovine serum (FBS) supplemented withpyruvate. Aliquots of 10⁴ cells in 100 μL were used in subsequentadhesion studies.

[0134] The cells were examined after plating using an inverted,phase-contrast microscope. At selected time points up to 4 days afterinitial seeding, the cells were rinsed three times in saline and fixedin buffered 10% formalin or 35% ethanol. In some cases, the cells werestained in situ with a 0.01 % aqueous solution of crystal violet. Theamount of attachment was scored visually or quantitated. To establishthe relative number of cells bound, crystal violet stained cells weredissolved in a solution of 70% ethanol containing 0.1% sodium dodecylsulfate and 0.38 M Tris and the absorbance monitored at 610 nm, usingthe methods of Scragg and Ferreira (Anal Biochem 198:80-5, 1991) andGrando et al. (Skin Pharmacol 6:135-47, 1993).

EXAMPLE 8

[0135] Attachment of Cells.

[0136] The cells of Example 7 were added to untreated polystyrene wells,wells coated with silyl-heparin as in Example 4, withsilyl-heparin-fibronectin as in Example 5, and with only fibronectin byincubation as described in Example 5. As shown in Table 2, C3H10T1/2fibroblasts, GS-9L gliosarcoma, Jurakt T cells, bovine aorta endothelial(BAE) cells and rat lymphocytes cultured in serum-containing medium onlow-attachment, nominally non-adherent polystyrene did not attach to thesubstrate. Similarly, these cells cultured on silyl-heparin alone didnot attach. When seeded following pre-treatment with bovine fibronectin,isolated C3H10T1/2 cells and isolated GS-9L cells were found attached.When cells were cultured on silyl-heparin-fibronectin, CH310T1/2, BAEand GS-9L cells rapidly attached and spread onto the substrate. Cellswere seeded at a concentration of 10⁴ cells/well in serum-containingmedium. TABLE 2 CELL ATTACHMENT SILYL- FI- HEPARIN- UN- SILYL- BRO-FIBRO- CELL TYPE TREATED HEPARIN NECTIN NECTIN GS-9L − − +/− ++++C3H10T1/2 − − +/− ++++ BAE − +/− − +++ Jurkat − − − − Lymphocytes − − −+/−

EXAMPLE 9

[0137] Adherence and Cell Spread.

[0138] Using the methods as described in Example 8, GS-9L, C3H10T1/2 andBAE cells were adhered by 1 hour after seeding onsilyl-heparin-fibronectin, and many of the cells demonstrated evidenceof spreading onto the substrate. By 2 hours nearly all of the cellsevidenced spreading. 10 The morphology of GS-9L, C3H10T1/2 and BAE cellsafter 24 hours on silyl-heparin-fibronectin were similar to cells grownon conventional tissue cultureware. The cells grew to confluency by 4days. Cells seeded in serum-free medium attached and spread although notas well as in serum-containing medium, and further did not grow. Noeffort was made to grow the cells in a defined- or serum-low mediumknown to support growth. Addition of up to 10 U of heparin to the medium15 of cells plated onto silyl-heparin-fibronectin coated plates did notinhibit the attachment or growth of C3H10T1/2. The results for GS-9L andC3H10T1/2 cells are shown in Table 3. TABLE 3 CHARACTERISTIC ATTACHMENTSPREADING GROWTH GS-9L CELLS Serum ++++ ++++ ++++ Serum-free +++ +++ −C3H10T1/2 CELLS Serum ++++ ++++ ++++ Serum-free +++ +++ −

EXAMPLE 10

[0139] Use on Variety of Substrates.

[0140] The general applicability of silyl-heparin-fibronectin complexwas evaluated by applying this coating using the methods of Examples 4and 5 to a variety of surfaces, including polystyrene,polylactide:polyglycolide, polycaprolactone, polyurethane and stainlesssteel. As in Example 9 above, C3H10T1/2 cells did not attach topolystyrene. C3H10T1/2 cells also did not attach to polyurethane. Theydid, however, bind moderately well to polylactide:polyglycolide and topolycaprolactone, and very well to stainless steel. Regardless of thesubstrate, coating with silyl-heparin essentially eliminated cellattachment. The inhibition of cell attachment was noted even on surfacesknown to support cell growth, such as stainless steel. Even when thecells were culture for up to 4 days, no cell attachment was evident.Treating the plates with heparin alone did not inhibit attachment.Fibronectin alone did not significantly support cell attachment topolystyrene. It did, however, improve the attachment of cells topolyurethane and polylactide:polyglycolide, and to a lesser extentpolycaprolactone. Silyl-heparin-fibronectin increased the cell densityfor all substrates relative to the uncoated surfaces, and increased thecell density on polycaprolactone by about 2-fold. The results aresummarized in Table 4. TABLE 4 C3H10T1/2 CELL ATTACHMENT SILYL- FI-HEPARIN- UN- SILYL- BRO- FIBRO- SURFACE TREATED HEPARIN NECTIN NECTINPolystyrene − − −/+ ++++ Polyurethane − − ++++ ++++ Polyactide: ++ −++++ ++++ polyglycolide Stainless steel* ++++ − ++++ ++++Polycaprolactone* +/− +/− ++ ++++

[0141] In related studies, heparin-specific assays and diemethylene bluedye uptake were used to establish that silyl-heparin complexes could bebound to a variety of materials including polystyrene, hydrogel-coatedpolystyrene, polycarbonate, polyurethane, poly caprolactone,polyvinylchloride, stainless steel and titanium. The loading efficiencyof silyl-heparin complexes varied by substrate material, but wasgenerally in the range of 10-40 mlU of heparin per cm².

EXAMPLE 11

[0142] Determination of Optimal Concentrations of Fibronectin andSilyl-Heparin.

[0143] The optimal amount of fibronectin and silyl-heparin to supportattachment of C3H10T1/2 cells was determined by cross-titration, asshown on FIG. 1. The EC₅₀ for fibronectin was approximately 0.5 μg/welland that of s-heparin about 8.7 μg/well. The cell attachment curve forthe fibronectin dilution had a rapid fall-off, suggestive of a thresholdeffect. The fall-off for the silyl-heparin was more gradual. The curveof the relative amount of silyl-heparin on the substrate followingserial dilution generally followed the cell attachment curve. The curveof the relative amount of fibronectin bound to silyl-heparin did notmirror the cell attachment curve.

[0144] In FIG. 1, fibronectin (

) or silyl-heparin (

) were titrated to determine the quantity needed to support attachmentof C3H10T1/2 cells. All dilutions were performed in quadruplicate inwells of 96-well plates of low-attachment polystyrene. For titration offibronectin, silyl-heparin was applied at 30 μg/well in 70% ethanol asin Example 4. After rinsing, fibronectin was applied in doublingdilutions of phosphate buffered saline starting from 4 μg/well. Fortitration of silyl-heparin, silyl-heparin was applied in doublingdilutions in 70% ethanol starting from silyl-heparin concentrations of30 μg/well. Fibronectin was then applied in saline at 4 μg/well. Cellswere at a concentration of 10⁴ cells/well in growth medium. For bothexperiments, the cells were allowed 24 hours to attach and then wererinsed and fixed in buffered formalin. The cells were stained for 5minutes with 0.01% aqueous crystal violet and the absorbance at 610 nmdetermined.

[0145]FIG. 2 shows the relative amounts of bound heparin and fibronectinin doubling dilutions to attached cell density. In FIG. 2A, the relativeamount of bound heparin (▪) was compared to subsequent attached celldensity following complexation with fibronectin () (4 μg/well). In FIG.2 B, the relative amount of bound fibronectin (FN) to wells coated withsilyl-heparin (300 μg/well) was compared to subsequent cell densityobtained after fibronectin complexation. Cell density was expressed asthe percent maximal absorbance of crystal violet. Heparin was detectedusing an enzyme-linked assay while fibronectin was detectedimmunochemically.

EXAMPLE 12

[0146] Use of Other Adhesive Molecules.

[0147] Laminin was attached to the silyl-heparin coated contactingsurfaces of Example 4 byn incubation in an aqueous 0.9% saline solutioncontaining 20 μg/mL intact murine laminin. After 30 minutes the unboundlaminin was removed by rinsing. In studies similar to those in Example8, C3H10T1/2 cells attached to surfaces coated with thesilyl-heparin-laminin complex.

EXAMPLE 13

[0148] Use of Different Silyl-Heparin-Adhesive Molecules.

[0149] Silyl-heparin complexes were evaluated for ability to bind toseveral adhesion molecules as shown in Table 5. Silyl-heparin complexesbound fibrinogen and a number of other adhesion molecules including typeIV collagen and fibronectin. Attachment of C3H10T1/2 cells after twohours to wells of 96-well, hydrogel-polystyrene plates coated withsilyl-heparin complexes and the listed adhesive molecule was detected bystaining with crystal violet. All proteins were used at a concentrationof 100 μg/ml in buffered saline. Serum was used without dilution.Statistical significance was assessed relative to the values frombuffered saline using Student's t-test, with * indicating p<0.05, **indicating p<0.01 and *** indicating p<0.001. TABLE 5 Adhesive MoleculeAverage ± S.E. Control (saline) 0.05 ± 0.03 albumin 0.08 ± 0.00fibrinogen  1.63 ± 0.17** fibronectin  1.00 ± 0.10** gelatin 0.15 ± 0.05Matrigel  1.70 ± 0.42* poly-l-ornithine  0.92 ± 0.28* poly-l-lysine  1.18 ± 0.06*** serum 0.16 ± 0.02 transferrin 0.15 ± 0.03 type VIcollagen   2.01 ± 0.08***

EXAMPLE 14

[0150] Preparation of Silyl-Heparin Complexes for use with Growth FactorMolecules.

[0151] Silyl-Heparin was prepared as generally disclosed in Example 1,with 3 silyl units per silyl moiety. The average number of silylmoieties per molecule of heparin was estimated based on a comparison ofthe molar ratios of the hydrolyzed prosthetic unitbenzyl-tri(dimethylsilyl-methyl)-OH and heparin as determined by use ofdimethyl methylene blue. Heparin was detected using acommercially-available enzyme-linked assay that measures theheparin-induced inhibition of antithrombin/factor Xa as measured with afactor Xa specific chromogenic substrate. The silyl-heparin complex wasused in a coating solution at 1% solution (w/v) in 60% aqueous ethanol.Contacting surfaces were coated with silyl-heparin for 15 minutes at 37°C. The wells were rinsed extensively in water, air-dried, and storeduntil use.

EXAMPLE 15

[0152] FGF Binding by Silyl-Heparin Complexes and Heparin.

[0153] Binding of bFGF to heparin agarose and various silyl-heparincomplexes of Example 14 were compared. Heparin agarose was rinsed inphosphate buffered saline and equilibrated in PBS containing 1% bovineserum albumin (PBS/BSA). Aliquots were added to microfuge tubes and thevolume adjusted to 0.5 mL with 0.5 mL of PBS/BSA. An additional 0.5 mLof Dulbecco's modified Eagle's medium containing 10% FBS containingheparin or various silyl-heparin complexes was added. An aliquot of 5 μLof bFGF (50 ng/mL) was added and the solutions incubated for 30 minuteswith mixing. The unbound bFGF was removed by multiple centrifugationsand rinsing in PBS. Rabbit anti-bFGF (1:500) was added in 0.5 mL PBS/BSAand the solution incubated with mixing for 1 hour. After rinsing donkeyanti-rabbit (1:500) in PBS/BSA was added and incubated 1 hour. Afterrinsing and transfer to new vials, chromogen (ABTS One-Step, PierceChemical Co.) was added and the reaction developed and subsequently readat 595 nm. The inhibition of binding of bFGF to heparin-agarose was usedto compare silyl-heparin complexes with between 5 and 20 silyl moietiesper heparin molecule with heparin not bound to silyl moieties. Theheparin used in this assay was the same as was used in the synthesis ofthe silyl-heparins. As shown in FIG. 3, each silyl heparin complexcomposition provided essentially the same EC₅₀ as unmodified heparinwhen used on equal weight basis.

EXAMPLE 16

[0154] FGF Concentration Binding by Silyl-Heparin Complexes.

[0155] Wells of 96 well polystyrene microtiter plates, with a wellsurface area of 0.32 cm², were coated with 30 μl of silyl heparin for 15minutes at 37° C. After rinsing and air-drying, bFGF (Sigma ChemicalCo.) in PBS/BSA was added in 150 μL aliquots in doubling dilutions. Theplate was incubated at 37° C. for 1 hour and rinsed 5 times in PBS. Analiquot 150 μl of PBS/BSA containing 1:500 rabbit anti-FGF antibodieswere added, incubated for 1 hour and rinsed. In some cases an irrelevantrabbit antibody was used at a similar concentration (rabbitanti-TGF-receptor II, Santa Cruz Biotechnology). An aliquot of PBS/BSAcontaining 1:500 HRPO-conjugated donkey anti-rabbit IgG added, and afterincubation and rinsing ABTS-chromogen added. All antibody solutions werefiltered through 0.2 micron filters prior to use. The absorbance wasmonitored at 405 nm. As shown in FIG. 4A and 4B, bFGF was detected overconcentrations from as low as 20 ng to as high as 750 ng. Colordevelopment was linear at high concentrations, indicating that underconditions of the assay the upper saturation limit of the silyl-heparincomplex for bFGF had not been reached. When anti-TGF-II (irrelevantantibody directed against the transforming growth factor receptor) wasused instead of anti-FGF and at the same anti-dilutions, no bFGF wasdetected. When TGF-β, at a concentration of 40 ng was used as the growthfactor molecule, no activity was detected with anti-bFGF antibodies.

EXAMPLE 17

[0156] Effect of Adhesive Molecules on FGF Binding.

[0157] Murine type IV collagen, human fibrinogen and bovine fibronectinisolated from plasma were bound to silyl-heparin complexes bound towells of polystyrene microtiter plates, at a concentration of 100 μg/mLin PBS/BSA. 100 μL of a selected adhesive molecule was added to eachwell, with incubation for 1 hour at 37° C. After extensive washing, bFGFin PBS/BSA in 150 μL aliquots was added to each well. The plate wasincubated at 37° C. for 1 hour and assayed as in Example 16 usingdilutions of 1:200 and 1:500 for the primary and secondary antibody,respectively. As shown in FIG. 5, fibronectin significantly decreasedthe binding of bFGF to the silyl-heparin-adhesive molecule complex, withless significant decrease in binding with type IV collagen andfibrinogen.

EXAMPLE 18

[0158] Capillary Tube Formation Using Silyl-Heparin-Growth FactorComplexes.

[0159] Low attachment 96 well plates were coated with saline,silyl-heparin complex, silyl-heparin-fibrinogen complex orsilyl-heparin-bFGF complex. Bovine aorta endothelial cells (5×10³ cells)were seeded into each well in DMEM containing 10% FBS. The cells wereallowed to grow for 4 days and the cultures examined by phase contrastmicroscopy. Bovine aorta endothelial cells underwent morphologicalchanges consistent with the formation of capillary tube formation whencultured on a contacting surface coated withsilyl-heparin-fibrinogen-bFGF. Tubes did not form when the cells werecultured in wells coated with silyl-heparin, silyl-heparin-fibrinogen,fibrinogen, fibrinogen and bFGF, or bFGF. On tubes coated withsilyl-heparin-fibrinogen the cells formed essentially confluentmonolayers.

EXAMPLE 19

[0160] Silyl-Heparin-FGF Complex Disassociation and Biological Activity.

[0161] Six-well cluster plates were seeded with 5×10⁴ bovine aortaendothelial cells. Cell culture insert membranes (25 mm diameter) with 3micron pores in the bottom were placed in each well. Suture material wascoated with silyl-heparin-bFGF or silyl-heparin. 10 cm of suturematerial, either coated or uncoated, was placed in each well and mediumadded sufficient to cover the suture (3 mL total). One set of insertscontaining an uncoated suture was spiked with 50 ng of bFGF by addingthe bFGF directly to the medium and was used as a positive control. Thecells were allowed to grow for 4 days, and were then fixed and stainedwith crystal violet. Thereafter, the stain was eluted in aqueousmethanol and 0.4% SDS and monitored at 595 nm. bFGF from thesilyl-heparin-bFGF coated suture was able to transit the separatingpermeable 3 micron membrane and act regionally to stimulate the growthof endothelial cells at the end of 4 days, as shown in FIG. 6. The cellnumbers increased in wells containing suture material coated withsilyl-heparin-bFGF complex and separated by the membrane to a similarextent as observed in control cultures spiked with soluble bFGF. Suturematerial coated with only silyl-heparin complex did not result in anincrease in cell number.

EXAMPLE 20

[0162] Sutures Coated with Silyl-Heparin-bFGF Implanted in Muscle.

[0163] Vicryl suture material was coated using a solution containingsilyl-heparin complex or a solution containing silyl-heparin complexfollowed by immersion in bFGF as described above. The suture materialwas then rinsed extensively, air-dried, mounted on 16 gage needles andstored until use, typically overnight. Under anesthesia, the sutureswere passed through the thigh muscle of adult rats and secured withknots and surgical clips. After two weeks the animals were euthanizedand the sutured area removed, fixed in formalin, and processed bystandard histological methods. At two weeks, sutures coated withsilyl-heparin-bFGF complex had a marked increase in cellularity in thearea surrounding the implant compared to both uncoated control suturesand sutures coated with silyl-heparin complex. As shown in FIG. 7, thediameter of granulation tissue surrounding the suture coated withsilyl-heparin-bFGF complex was substantially larger than either thecontrol suture or silyl-heparin complex coated sutures. It was alsoobserved that tissue surrounding the suture material coated withsilyl-heparin complex tended to have a smaller area of granulation thanthat of the control suture.

EXAMPLE 21

[0164] Detection of Vascular Endothelial Growth Factor (VEGF) byImmunoassay.

[0165] All antibody solutions were filtered through 0.2 micron filtersprior to use and prepared in phosphate buffered saline (PBS) containing1% bovine serum albumin (BSA). Wells of 96 well polystyrene microtiterplates were coated with 30 μL of silyl heparin for 15 minutes at 37° C.The wells were rinsed extensively in water and air-dried. The wells wereblocked by adding 250 μL of PBS containing 1 % BSA for 30 minutes. Afterremoving the blocking solution, VEGF in PBS/BSA was added in 100 μL andat the appropriate concentration. The plate was incubated at 37° C. for1 hour and rinsed 5 times in water. An aliquot of PBS/BSA containingrabbit anti-VEGF antibodies (1:125) was added and the plate incubatedfor one hour. After rinsing in water, an aliquot of PBS/BSA containinghorseradish peroxidase-conjugated anti-rabbit IgG (1:250) was added andincubated for one hour. After rinsing, 200 μL of chromogen(2,2′-azinobis 3-ethylbenzothiazoline-6-sulfinic acid) from acommercially available kit was added, the color developed, and thereaction stopped by adding 50 μL of aqueous 2% sodium dodecyl sulfate(SDS). The absorbance was monitored at 405 nm. The results, as shown inFIG. 8, demonstrate that VEGF bound to silyl-heparin and the binding waslinear.

EXAMPLE 22

[0166] Effect of varying “x” and “n” on Disassociation.

[0167] Silyl-heparin complexes of Formula III were synthesized byvarying a) the silyl chain-length in the silyl moiety (“n”), and b) thenumber of silyl moieties per heparin molecule (“x”). The resident timeof the silyl-heparin bound to stainless steel varied, with silyl-heparincomplexes where n =4 having the longest resident time. Varying thenumber of silyl moieties per heparin molecule, studied over the rangefrom x=2 to x=20 also affected the resident time of the silyl-heparincomplex on stainless steel, with the longest resident time observedwhere x=4 to x=6.

[0168] The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

[0169] Although the invention has been described in detail withparticular reference to these preferred embodiments, other embodimentscan achieve the same results. Variations and modifications of thepresent invention will be obvious to those skilled in the art and it isintended to cover in the appended claims all such modifications andequivalents. The entire disclosures of all references, applications,patents, and publications cited above are hereby incorporated byreference.

1 2 1 5 PRT Artificial laminin basement membrane derived peptide 1 IleLys Val Ala Val 1 5 2 5 PRT Artificial laminin basement membrane derivedpeptide 2 Tyr Ile Gly Ser Arg 1 5

What is claimed is:
 1. A coating composition for contacting surfaces ofmedical device, said composition comprising a molecule of Formula I:

wherein R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group, each R₂ isindependently selected from the group consisting of C₁₋₁₈ alkyl andC₆₋₃₂ aryl R₃ is N or O, n is a number from 1 to 10, and x is a numberfrom 1 to about 30, bound to a heparin-activity molecule via a covalentbond, thereby forming a silyl-heparin covalent complex, with a bioactivemolecule directly bound to the heparin-activity molecule.
 2. Thecomposition according to claim 1, wherein the silyl-heparin covalentcomplex binds to the contacting surfaces via hydrophobic bondinginteractions.
 3. The composition according to claim 1, wherein thesilyl-heparin covalent complex has a disassociation rate from thecontacting surface determined by the value of n and x.
 4. Thecomposition according to claim 2, wherein the heparin-activity moleculeis heparin, heparan sulfate, hyaluronic acid, dextran, dextran sulfate,chondroitin sulfate, dermatan sulfate, or a molecule including a mixtureof variably sulfated polysaccharide chains composed of repeating unitsof D-glucosamine and either L-iduronic or D-glucuronic acids, salts ofany of the foregoing and derivatives of any of the foregoing.
 5. Thecomposition according to claim 1, wherein said bioactive molecule is anadhesive molecule, a growth factor molecule or a therapeutic molecule.6. The composition according to claim 1, wherein said bioactive moleculeis directly bound to the heparin-activity molecule by affinitycomplexation.
 7. The composition according to claim 1, wherein thesilyl-heparin covalent complex comprises[benzyl-bis(dimethylsilylmethyl)]-(N-heparinyl)-carbamate or[benzyl-tris(dimethylsilylmethyl)]-(N-heparinyl)-carbamate.
 8. Thecomposition according to claim 5, wherein the adhesive molecule isfibronectin, laminin, vitronectin, thrombospondin, gelatin, polylysine,polyornithine, peptide polymers containing adhesive sequences andheparin binding sequences, sulfated complex carbohydrates, dextransulfate, growth hormones, cytokines, lectins, or peptidic polymersthereof.
 9. The composition according to claim 5, wherein the growthfactor molecule is fibroblast growth factor, platelet-derived growthfactor, vascular endothelial growth factor, hepatocyte growth factor,placental growth factor, insulin-like growth factor, nerve growthfactors and neurotrophins, heparin-binding epidermal growth factor,transforming growth factor-β, bone morphogenetic protein 2, osteogenicprotein 1 or keratinocyte growth factor.
 10. A medical device with atleast one contacting surface for contacting bodily fluids, the surfacehaving coated thereon a coating composition comprising a molecule ofFormula I:

wherein R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group, each R₂ isindependently selected from the group consisting of C₁₋₁₈ alkyl andC₆₋₃₂ aryl, R₃ is N or O, n is a number from 1 to 10, and x is a numberfrom 1 to about 30, directly bound to a heparin-activity molecule viacovalent bonding, thereby forming a silyl-heparin covalent complex, witha bioactive molecule directly bound to the heparin-activity molecule.11. The device according to claim 10, wherein the silyl-heparin covalentcomplex binds to the surface via hydrophobic bonding interactions. 12.The device according to claim 10, wherein the silyl-heparin covalentcomplex has a disassociation rate from the surface determined by thevalue of n and x.
 13. The device according to claim 10, wherein theheparin-activity molecule is heparin, heparan sulfate, hyaluronic acid,dextran, dextran sulfate, chondroitin sulfate, dermatan sulfate, or amolecule including a mixture of variably sulfated polysaccharide chainscomposed of repeating units of D-glucosamine and either L-iduronic orD-glucuronic acids, salts of any of the foregoing and derivatives of anyof the foregoing.
 14. The device according to claim 10, wherein saidbioactive molecule is an adhesive molecule, a growth factor molecule ora therapeutic molecule.
 15. The device according to claim 10, whereinsaid bioactive molecule is directly bound to the heparin-activitymolecule by affinity complexation.
 16. The device according to claim 10,wherein the silyl-heparin covalent complex comprises[benzyl-bis(dimethylsilylmethyl)]-(N-heparinyl)-carbamate or[benzyl-tris(dimethylsilylmethyl)]-(N-heparinyl)-carbamate.
 17. Thedevice according to claim 14, wherein the adhesive molecule isfibronectin, laminin, vitronectin, thrombospondin, gelatin, polylysine,polyornithine, peptide polymers containing adhesive sequences andheparin binding sequences, sulfated complex carbohydrates, dextransulfate, growth hormones, cytokines, lectins, or peptidic polymersthereof.
 18. The device according to claim 14, wherein the bioactivemolecule is an adhesive molecule, whereby the contacting surface isnon-thrombogenic and promotes cellular adhesion.
 19. The deviceaccording to claim 14, wherein the growth factor molecule is fibroblastgrowth factor, platelet-derived growth factor, vascular endothelialgrowth factor, hepatocyte growth factor, placental growth factor,insulin-like growth factor, nerve growth factors and neurotrophins,heparin-binding epidermal growth factor, transforming growth factor-β,bone morphogenetic protein 2, osteogenic protein 1 or keratinocytegrowth factor.
 20. The device according to claim 10, wherein the deviceis a blood gas exchange device, blood filter, artificial blood vessel,artificial valve, prosthetic, blood shunt, catheter, bone replacement,cartilage replacement, suture, graft, catheter or nerve growth guide.21. A method for coating a contacting surface of a medical device with abioactive coating composition, comprising: providing a hydrophobic silylmoiety of Formula I

wherein R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group, each R₂ isindependently selected from the group consisting of C₁₋₁₈ alkyl andC₆₋₃₂ aryl, R₃ is N or O, and n is a number from 1 to 10 binding saidsilyl moiety to a heparin-activity molecule via covalent bonding,wherein x is from 1 to about 30 for each heparin-activity molecule,thereby forming a silyl-heparin complex, attaching the silyl-heparincomplex to the contacting surface by hydrophobic interaction, andattaching a bioactive molecule to the heparin-activity molecule.
 22. Themethod of claim 21, wherein the silyl-heparin complex has adisassociation rate from the contacting surface determined by the valueof n and x.
 23. The method of claim 21, wherein the heparin-activitymolecule is heparin, heparan sulfate, hyaluronic acid, dextran, dextransulfate, chondroitin sulfate, dermatan sulfate, or a molecule includinga mixture of variably sulfated polysaccharide chains composed ofrepeating units of D-glucosamine and either L-iduronic or D-glucuronicacids, salts of any of the foregoing and derivatives of any of theforegoing.
 24. The method of claim 21, wherein the bioactive molecule isan adhesive molecule, a growth factor molecule or a therapeuticmolecule.
 25. The method of claim 21, wherein the bioactive molecule isattached to the heparin-activity molecule by affinity complexation. 26.The method of claim 21, wherein the silyl-heparin complex comprises[benzyl-bis(dimethylsilylmethyl)]-(N-heparinyl)-carbamate or[benzyl-tris(dimethylsilylmethyl)]-(N-heparinyl)-carbamate.
 27. Themethod of claim 24, wherein the adhesive molecule is fibronectin,laminin, vitronectin, thrombospondin, gelatin, polylysine,polyornithine, peptide polymers containing adhesive sequences andheparin binding sequences, sulfated complex carbohydrates, dextransulfate, growth hormones, cytokines, lectins, or peptidic polymersthereof.
 28. The method of claim 21, wherein the bioactive molecule isan adhesive molecule, whereby the contacting surface is non-thrombogenicand promotes cellular adhesion.
 29. The method of claim 24, wherein thegrowth factor molecule is fibroblast growth factor, platelet-derivedgrowth factor, vascular endothelial growth factor, hepatocyte growthfactor, placental growth factor, insulin-like growth factor, nervegrowth factors and neurotrophins, heparin-binding epidermal growthfactor, transforming growth factor-β, bone morphogenetic protein 2,osteogenic protein 1 or keratinocyte growth factor.